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Simple Summary: The interactions between parasitoids and their host have always attracted much attention, and parasitoids manipulate the host physiology to benefit the survival and development of their offspring. It is critical to analyze these physiological regulatory mechanisms. In this study, using RNA-sequencing analysis, we clarified the effects of parasitization on host gene expression of Microplitis manilae, a dominant larval parasitoid of Spodoptera frugiperda. Several differentially expressed genes were identified in S. frugiperda larvae at 2 h, 24 h, and 48 h post-parasitization. Among these, many genes were implicated in host metabolism and immunity. Further qPCR results involving 20 differentially expressed metabolism, and immune-related genes were consistent with the expression profiles of the RNA-seq data, which confirmed the physiological manipulation of M. manilae on host S. frugiperda. These results contribute to defining the molecular basis of wasps' successful parasitization and facilitate their application. It has been extensively found that parasitoids manipulate host physiology to benefit the survival and development of their offspring. However, the underlying regulatory mechanisms have not received much attention. To reveal the effects of parasitization of the larval solitary endoparasitoid Microplitis manilae (Hymenoptera: Braconidae) on host Spodoptera frugiperda (Lepidoptera: Noctuidae), one of the most destructive agricultural pests in China, deep-sequencing-based transcriptome analysis was conducted to compare the host gene expression levels after 2 h, 24 h, and 48 h parasitization. A total of 1861, 962, and 108 differentially expressed genes (DEGs) were obtained from the S. frugiperda larvae at 2 h, 24 h, and 48 h post-parasitization, respectively, compared with unparasitized controls. The changes in host gene expressions were most likely caused by the injection of wasp parasitic factors, including PDVs, that were injected along with the eggs during oviposition. Based on the functional annotations in GO and KEGG databases, we revealed that most DEGs were implicated in host metabolism and immunity. Further analysis of the common DEGs in three comparisons between the unparasitized and parasitized groups identified four genes, including one unknown and three prophenoloxidase (PPO) genes. Moreover, 46 and 7 common DEGs involved in host metabolism and immunity were identified at two or three time points after parasitization, respectively. Among these, most DEGs showed increased expressions at 2 h post-wasp parasitization while exhibiting significantly decreased expression levels at 24 h post-parasitization, demonstrating the expression regulations of M. manilae parasitization on host metabolism and immune-related genes. Further qPCR verification in 20 randomly selected DEGs confirmed the accuracy and reproducibility of the gene expression profiles generated from RNA-seq. This study reveals the molecular regulatory network about how host insects respond to wasp parasitism, laying a solid foundation for revealing the physiological manipulation of wasp parasitization on host insects, which facilitates the development of biological control practices for parasitoids. [ABSTRACT FROM AUTHOR]